Protocol for Running Bulk Species-Mixing RNA-Seq using ChronoSeq or Dropseq beads

Solutions and Buffers to Make

• 1M DTT:
  • Dissolve 1.55g solid DTT in 10ml of Deionized Water. Hazardous Chemical Read SDS. Work in Fume Hood

More economical to make yourself from Solid DTT. Make 50ml or more at a time and store in 10ml Aliquots for upto an year. DTT is not stable at room temperature. Make this solution quickly and store at -20°C, unless you plan to use it immediately to make another Buffer.

• 5X RT Buffer:
  • 250mM Tris-HCl (pH 8.3 at 25°C)
  • 375mM KCl
  • 15mM MgCl₂
  • 50mM DTT

More economical to make yourself for large volumes. Make 100ml of more at a time and store in 11ml Aliquots at -20°C for upto an year. DTT is not stable at room temperature. Make this solution quickly and freeze.

Preparation a Day Before

• Turn on the UV for the Cell culture hood and wait 15 minutes.
• Aseptically prepare 4 eppendorf tubes with 10μl for each bead type, condition or timepoint the day before. Bead concentration should be 450beads/μl:
  • Spin down the beads in the Tube in which your beads are stored.
  • Vortex this Tube at Maximum speed for 3 seconds to make sure the beads are evenly suspended before transfering them.
  • There should be 4 eppies for each type of bead/timepoint/condition.
  • Two eppies should be labeled with H(Human) and Name of Condition/Bead type/Timepoint.
  • The remaining two should be labeled with M(Mouse) and Name of Condition/Bead type/Timepoint.
  • For example if there are two conditions there should be 8 total eppies each with 10μl of beads.

Mix Beads with Cell Lines

• Prepare enough 40μm filtered 6X SSC Buffer for the experiment. You will need 1ml of 6X SSC buffer per eppie. Keep this buffer at room temperature.
• Prepare enough 1.25X RT buffer from 5X Stock for the experiment. You will need 2ml of 1.25X RT buffer per eppie. Put this buffer on Ice.
• Using the Protocol for Preparing Cells make Cell Suspensions for 3T3 and HEK293 at 600 cells/μl for both. Do not mix the cell lines, prepare a separate tube with 1ml each.
• Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Add the Cell suspension to the beads and mix well:
  • There should be four eppies for each condition. Two for Human Cells and Two for Mouse Cells.
  • Mix 8μl of Mouse Cells(3T3) for the M(Mouse) eppies. Pipette up and down several times to mix well.
  • Then quickly put these eppies on ice.
  • Mix 8μl of Human Cells(HEK293) for the H(Human) eppies. Pipette up and down several times to mix well.
  • Then quikcly put these eppies on ice.

Wash Beads with 6X SSC and 1.25X RT Buffer

• Wash each eppie once with 1ml of 6X SSC and twice with 1ml of 1.25X RT buffer:
  • Add 1ml of room temperature 6X SSC buffer. Vortex the beads as full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Carefully remove 900μl of Liquid without disturbing the beads.
  • Add 1ml of 1.25X RT buffer. Vortex the beads at full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Carefully remove 1ml of Liquid without disturbing the beads.
  • Add 1ml of 1.25X RT buffer. Vortex the beads at full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Label a new eppies with the Condition/Bead-Type/Timepoint and date of the experiment. For example, if there are two Conditions/Bead-Types/Timepoints then you will need two new eppies.
• Carefully remove 900μl of 1.25X RT buffer from each eppie without disturbing the beads.
• Now combine all 4 Eppies(Human+Mouse) for a single Condition/Bead Type/Timepoint into the new Eppies.
• Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Follow the Library Preparation Protocol from this point onwards.

Protocol for Running Bulk Time-Series RNA-Seq using ChronoSeq beads

Solutions and Buffers to Make

• 1M DTT:
  • Dissolve 1.55g solid DTT in 10ml of Deionized Water. Hazardous Chemical Read SDS. Work in Fume Hood

More economical to make yourself from Solid DTT. Make 50ml or more at a time and store in 10ml Aliquots for upto an year. DTT is not stable at room temperature. Make this solution quickly and store at -20°C, unless you plan to use it immediately to make another Buffer.

• 5X RT Buffer:
  • 250mM Tris-HCl (pH 8.3 at 25°C)
  • 375mM KCl
  • 15mM MgCl₂
  • 50mM DTT

More economical to make yourself for large volumes. Make 100ml of more at a time and store in 11ml Aliquots at -20°C for upto an year. DTT is not stable at room temperature. Make this solution quickly and freeze.

Preparation a Day Before

• Prepare 12 eppendorf tubes with 10μl for each bead timepoint/Seq Number the day before. Bead concentration should be 450beads/μl:
  • Spin down the beads in the Tube in which your beads are stored.
  • Vortex this Tube at Maximum speed for 3 seconds to make sure the beads are evenly suspended before transfering them.
  • There should be 12 eppies for each timepoint/Seq Number (SEQ1-SEQ12).

Mix Beads with Cell Suspension Sample

• Prepare enough 40μm filtered 6X SSC Buffer for the experiment. You will need 1ml of 6X SSC buffer per eppie. Keep this buffer at room temperature.
• Prepare enough 1.25X RT buffer from 5X Stock for the experiment. You will need 2ml of 1.25X RT buffer per eppie. Put this buffer on Ice.
• Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Add the Cell suspension @600cells/μl to the beads and mix well:
• Immediately after the Cell Suspension Sample is deposited into a 15ml Tube you can take a 8μl sample and mix it with your ChronoSeq beads.
  • Mix the Sample with its Corresponsing Seq Number for ChronoSeq beads.
  • For example for Cell Sample number 1, mix with Seq 1. For Cell Sample number 5 mix with Seq 5 etc..
  • Pipette up and down several times to mix well.
• Immediately after mixing put the beads on Ice. Keep the beads on Ice till all the Cell Samples have been collected.
  • Put the Tubes down deep into the ice.
  • Compress the Ice to make sure the bottom of the tubes remain in constant contact with the ice.
• Keep the Cell Suspension Samples in separate rack at room temperature.

Wash ChronoSeq Beads with 6X SSC and 1.25X RT Buffer

• Wash each eppie once with 1ml of 6X SSC and twice with 1ml of 1.25X RT buffer:
  • Add 1ml of room temperature 6X SSC buffer. Vortex the beads as full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Carefully remove 900μl of Liquid without disturbing the beads.
  • Add 1ml of 1.25X RT buffer. Vortex the beads at full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Carefully remove 1ml of Liquid without disturbing the beads.
  • Add 1ml of 1.25X RT buffer. Vortex the beads at full speed for 2 seconds.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Label a new eppie with a Brief Description and date of the Experiment.
• Carefully Remove 1000μl of 1.25X RT buffer from each eppie without disturbing the beads.
• Now combine all timepoints/seq numbers into the new Eppie using a P200.
• Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
• Follow the Library Preparation Protocol from this point onwards.